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Vol. 59, No 4/2012
693–696
on-line at: www.actabp.pl
Regular paper
Quantitative estimation of lysosomal storage in
mucopolysaccharidoses by electron microscopy analysis
Magdalena Narajczyk1*, Marta Moskot2 and Aleksandra Konieczna2
Laboratory of Electron Microscopy; 2Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland
1
Mucopolysaccharidoses (MPS) are severe inherited metabolic disorders caused by storage of glycosaminoglycans
(GAGs). The level of accumulated GAGs is an important
parameter in assessment of the severity of the disease
and the efficacy of treatment; unfortunately, biochemical
methods are often unreliable and only semi-quantitative.
Therefore, finding other methods for estimation of GAG
levels and/or assessment of the efficacy of applied therapy is very important. Although monitoring of GAG levels
during therapy is crucial, in this work it is proposed that
analysis of the ultrastructure of MPS cells by electron microscopic techniques can be considered as an alternative
and reliable method for assessment of lysosomal storage. The number of complex lysosomal structures was
found to be significantly higher in MPS cells relative to
controls, while it decreased significantly in response to
either enzyme replacement therapy or substrate reduction therapy. Thus, this parameter, easily assessed by
electron microscopy, appears to correspond to the physiological state of MPS cells.
Key words: mucopolysaccharidoses, glycosaminoglycans, transmission electron microscopy
Received: 17 November, 2012; revised: 27 November, 2012;
accepted: 06 December, 2012; available on-line: 11 December, 2012
INTRODUCTION
Mucopolysaccharidoses (MPS) are rare, inherited lysosomal storage disorders caused by deficiencies in activities of lysosomal hydrolases (Neufeld & Muenzer, 2001).
In these diseases, defects in degradation of glycosaminoglycans (GAGs) are caused by mutations in genes coding
for these enzymes. This leads to accumulation of GAGs
in lysosomes, which compromises the functioning of
different tissues and organs and usually causes death in
childhood (Neufeld & Muenzer, 2001).
One of the most promising therapies of MPS is enzyme replacement therapy (ERT), which is now registered for three types of MPS (types I, II and VI). Unfortunately, this treatment is ineffective in managing neurological and behavioral symptoms of neuronopathic MPS
types I, II and VII, in which pathological changes occur
in the central nervous system (CNS), as the enzyme cannot cross the blood-brain barrier (Beck, 2007a, 2007b;
Węgrzyn et al., 2010). Another treatment mode is bone
marrow transplantation, which has been reported to be
efficient only if performed during the first two years of
life and only in MPS type I (Beck 2007a, 2007b). Recently, yet another possible therapy was proposed, called
gene expression-targeted isoflavone therapy (GET IT),
which is a specific kind of substrate reduction therapy
(SRT) and has been suggested to be useful in treatment
of various types of mucopolysachcaridoses and other lysosomal storage diseases (Piotrowska et al., 2006; Otomo
et al., 2012).
Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H1-benzopyran-4-one), a natural isoflavone, significantly
inhibits GAG synthesis and decreases GAG storage in
fibroblasts of patients suffering from different MPS and
mucolipidosis (ML) types (Piotrowska et al., 2006; Otomo
et al., 2012). Since genistein has been reported to cross
the blood-brain barrier (Tsai, 2005), it was proposed to
be useful in the treatment of neurological symptoms of
MPS and ML. Studies performed on animal models of
MPS II (Friso et al., 2010) and MPS IIIB (Malinowska
et al., 2010) showed a significant reduction in lysosomal
storage. Clinical studies with MPS III patients performed
with the use a genistein-rich isoflavone extract at doses
of 5 to 15 mg/kg/day showed a decrease in GAG levels, improvement of hair morphology, and behavior (Piotrowska et al., 2008, 2011; Delgadillo et al., 2011; Malinova et al., 2012, de Ruijter et al., 2012).
Despite the encouraging results of GET IT and reported clinical benefits for patients (Piotrowska et al.,
2008, 2011), some authors suggested a limited or even
negligible efficacy of this therapy (Delgadillo et al., 2011).
One of the causes of these discrepancies might be unprecise and semi-quantitative biochemical methods of
measuring GAG levels. This problem was signalled previously (Piotrowska et al., 2009) and concerns both biological human samples and the material from cell cultures.
One of alternative methods for GAG storage estimation is transmission electron microscopy (TEM) study.
In fact, this method has been used for over 45 years to
investigate pathology of MPS (Aleu et al., 1965; Lagunoff
& Gritzka, 1966), and TEM analyses were performed recently during tests with different possible medicines (Piotrowska et al., 2006; Jakóbkiewicz-Banecka et al., 2009;
Kloska et al., 2011; Dziedzic et al., 2012). The aim of
the present work is to assess whether TEM can be used
for quantitative estimation of lysosomal storage. To perform such an assessment, TEM analysis was performed
in an experimental system in which storage levels could
be easily predicted, i.e. in untreated MPS cells and after
various treatments.
*
e-mail: [email protected]
Abbreviations: MPS, mucopolysaccharidosis; TEM, transmission
electron microscopy; GAG, glycosaminoglycans; ERT, enzyme replacement therapy; SRT, substrat reduction therapy; GET IT, gene
expression-targeted isoflavone therapy; CNS, central nervous system; SEM, scaning electron microscopy; ML, mucolipidosis.
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M. Narajczyk and others
2012
Figure 1. Different lysosomal structures observed in fibroblasts.
Electron micrographs present: (a) lamellar lysosome (asterisk) and complex lysosomal structure (arrow) in the cell, magnification 21 000x;
(b) control (untreated wild type cells); (c) untreated MPS I cells; (d) MPS I cells treated with 30 μM genistein; (e) MPS I cells treated with
50% Aldurazyme and 30 μM genistein; (f) MPS I cells treated with 100% Aldurazyme; (g) untreated MPS IIIA cells; (h) MPS IIIA cells treated with 30 μM genistein; all micrographs except panel (a) were made at magnification 1650x.
MATERIALS AND METHODS
Cell lines and culture conditions. Fibroblast cell
lines obtained from MPS I and MPS IIIA patients (Memorial Children Hospital, Warsaw, Poland) were used
in experiments. Human Dermal Fibroblast adult line
(HDFa; Cascade Biologics, Portland, OR, USA) was
used as a healthy control line. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with 10% Fetal Bovine Serum (FBS) and 1 x Antibiotic
and Antimycotic Solution (all purchased from Sigma,
Germany) at 37°C in humidified 5% CO2 atmosphere.
Enzyme and genistein. Human recombinant α-liduronidase (Aldurazyme) was obtained from Genzyme
Co. (Cambridge, MA, USA). Genistein was purchased
from Sigma (Germany).
Electron microscopic studies. Cells were incubated in growth medium supplemented with appropriate
concentrations of tested compounds for 14 days. Cells
were fixed with 2.5% glutaraldehyde, followed by PBS
washing. Then, the cells were fixed with 1% osmium
tetroxide and 1% potassium hexacyanoferrate (III) (all
purchased from Sigma, Germany), which was followed
by ethanol dehydration. Ultrasections (approximately 40
nm) of cells embedded in Epon 812 resin (Fluka, Germany) were stained in lead citrate and uranyl acetate
(Herman-Antosiewicz et al., 2006), examined under a
transmission electron microscope (Philips CM100) and
photographed at magnification 1650x using iTEM program (Olympus Soft Imaging Solution). Each experiment
was blinded at the time of analysis and reporting. All
photographed fibroblasts had intact membrane and visible cells structures (nucleus, mitochondria, lysosomes).
At least 20 cells were examined and for each cell at least
5 random fields were assessed. Lamellar lysosomes and
complex lysosomal structures (Fig. 1a) were counted per
100 μm2 of cross-section of a cell.
RESULTS
The fibroblasts were cultured for 14 days in the absence or presence of different compounds. Following
cell preparation, their lysosomal structure was examined
under transmission electron microscope (Fig. 1).
Although various abnormal structures were observed,
complex lysosomal structures appeared to be the most
characteristic for MPS cells. When calculated per 100
µm2, the number of these structures correlated well
with the expected results of the treatment (the effects
of various treatments had been demonstrated previously
by other authors and using other methods, see Unger
et al., 1994; Piotrowska et al., 2006). Thus, while there
were many complex lysosomal structures in untreated
Vol. 59 Lysosomal storage in mucopolysaccharidoses by electron microscopy analysis
695
port, a TEM method is proposed
for assessment of lysosomal storage in MPS cells. Despite the fact
that TEM had been used previously in many studies on MPS, it
appeared important to develop a
procedure to estimate the level of
storage quantitatively.
The results presented in this
report suggest that determination
of the number of complex lysosomal structures per 100 µm2 of
a cell cross-section may be an
adequate measure of pathological state in MPS cells. The results
presented in Fig. 2 are compatible
with expectations of effects of
various treatments of MPS cells
on lysosomal storage, based on
previously published results where
Figure 2. Effects of genistein, Aldurazyme and their combination on the presence of comthis parameter was carefully inplex lysosomal structures in HDFa, MPS I and MPS IIIA fibroblasts.
The presented values are mean values from 20 analyzed cell (with 5 views of each cell). Ervestigated by methods (Unger et
ror bars indicate S.D.
al., 1994; Piotrowska et al., 2006).
Therefore, the use of TEM and
MPS I and MPS IIIA cells, treatment of MPS I cells
determination of the frequency of
with recombinant human α-L-iduronidase (Aldurazyme) complex lysosomal structures is suggested as a conveniat the dose recommended for the therapy (described as ent method for estimation of the level of lysosomal stor100% dose in this report) resulted in a drastic decrease age in MPS.
in this parameter (Fig. 2). Similarly, treatment of MPS
I and MPS IIIA cells with 30 µM genistein caused a Acknowledgements
smaller but significant decrease in the number of comThis research was supported by the University of
plex lysosomal structures (Fig. 2). A significant effect
was also observed when a combined treatment, 50% Gdańsk (grant no. 538-L107-0806-12) and was operated
dose of Aldurazyme and 30 µM genistein, was applied within the Foundation for Polish Science Team Pro(Fig. 2). These results strongly suggest that analysis of gramme co-financed by the EU European Regional Dethe frequency of complex lysosomal structures may be velopment Fund (grant no. TEAM/2008-2/7).
an adequate method for estimation of lysosomal storage
in MPS.
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The use of proper biomarkers in investigation of
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